Bovine DNA Research Articles

Interaction of superparamagnetic iron oxide nanoparticles with DNA and BSA

Background: Superparamagnetic iron oxide nanoparticles (SPION) are widely used in various biomedical technologies, in particular, as carriers for drug delivery to the target. Since SPION-drug complexes are planned to be used in vivo, it is necessary to find out if competitive binding of nanoparticles with biologically important macromolecules (nucleic acids and proteins) is possible. Objectives: To investigate the possibility of complexation of iron oxide nanoparticles with DNA and serum albumin. Materials and methods: Bare and sodium citrate coated SPION with different surface charges, bovine serum albumin (BSA) and calf thymus DNA were used. The complexes of SPION and macromolecules were precipitated by an external magnetic field. The research was carried out by spectrophotometry in visible and ultraviolet ranges. Results: To study SPION interactions with DNA and BSA, the spectra of supernatants of the binary systems were compared with the spectra of the corresponding control macromolecules solutions. In the DNA-SPION systems, a decrease in the DNA absorption is observed only for bare nanoparticles. Our estimation shows that the maximum possible concentration ratio of bound DNA to SPION is about 2.5×10-4 mol/g. The addition of sodium citrate coated SPION to the DNA solution does not cause any spectral changes of the supernatant. The interaction of BSA with SPION, coated with sodium citrate, leads to a slight increase in supernatant absorption compared with the one of the control protein solution. It can be caused by the fact that the resulting complexes are not precipitated by a magnetic field. No difference between the spectrum of the supernatant of BSA-bare SPION system and the control protein solution was observed. Conclusions: The obtained spectrophotometric results demonstrate the formation of complexes between DNA and bare iron oxide nanoparticles as well as between BSA and the nanoparticles, coated with sodium citrate. The maximum concentration ratio of bound DNA and bare SPION was obtained for the investigated system. It is necessary to take into account when SPION are used as carriers for drug delivery.

Insight into Rapid DNA-Specific Identification of Animal Origin Based on FTIR Analysis: A Case Study.

In this study, a methodology has been proposed to identify the origin of animal DNA, employing high throughput extension accessory Fourier transform infrared (HT-FTIR) spectroscopy coupled with chemometrics. Important discriminatory characteristics were identified in the FTIR spectral peaks of 51 standard DNA samples (25 from bovine and 26 from fish origins), including 1710, 1659, 1608, 1531, 1404, 1375, 1248, 1091, 1060, and 966 cm−1. In particular, the bands at 1708 and 1668 cm−1 were higher in fish DNA than in bovine DNA, while the reverse was true for the band at 1530 cm−1 was shown the opposite result. It was also found that the PO2− Vas/Vs ratio (1238/1094 cm−1) was significantly higher (p < 0.05) in bovine DNA than in fish DNA. These discriminatory characteristics were further revealed to be closely related to the base content and base sequences of different samples. Multivariate analyses, such as principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were conducted, and both the sensitivity and specificity values of PLS-DA model were one. This methodology has been further validated by 20 meat tissue samples (4 from bovine, 5 from ovine, 5 from porcine, and 6 from fish origins), and these were successfully differentiated. This case study demonstrated that FTIR spectroscopy coupled with PLS-DA discriminant model could provide a rapid, sensitive, and reliable approach for the identification of DNA of animal origin. This methodology could be widely applied in food, feed, forensic science, and archaeology studies.

Novel multiplex PCR-RFLP assay discriminates bovine, porcine and fish gelatin substitution in Asian pharmaceuticals capsule shells

ABSTRACTGelatin is widely used in pharmaceuticals as a protective coating, such as soft and hard capsule shells. However, the animal source of gelatin is a sensitive issue because certain gelatins such as porcine and bovine gelatins are not welcome in Halal, Kosher and Hindus’ consumer goods. Recently, we have documented DNA barcoding and multiplex PCR platforms for discriminating porcine, bovine and fish gelatins in various fish and confectionary products; but those assays were not self-authenticating and also not tested in highly refined pharmaceutical products. To address this knowledge gap, here we report a self-authenticating multiplex PCR-restriction fragment length polymorphism (RFLP) assay to identify animal sources of various gelatin in pharmaceutical capsules. Three different restriction enzymes, BsaAI, Hpy188I and BcoDI were used to yield distinctive RFLP patterns for gelatin-based bovine (26, 94 bp), fish (97, 198 bp) and porcine (17, 70 bp) DNA in control experiments. The specificity was cross-tested against 16 non-target species and the optimised assay was used to screen gelatin sources in 30 halal-branded pharmaceuticals capsule shells. Bovine and porcine DNA was found in 27 and 3 of the 30 different capsules products. The assay was suitable for detecting 0.1 to 0.01 ng total DNA extracted from pure and mixed gelatins. The study might be useful to authenticate and monitor halal, kosher, vegetarian and Hindu compliant pharmaceuticals, foods and cosmetics.

Síntesis, actividad antibacteriana e interacción del ADN con complejos de inclusión entre compuestos lantánidos y β-ciclodextrina

En este trabajo se han sintetizado complejos de lantánidos a partir de los derivados cloruros de La(III), Ce(III), Sm (III) e Yb(III) con ligandos cinamato, presentando coordinación bidentada entre el grupo carboxilo del ligando y el metal lantánido. Estos compuestos se utilizaron como huéspedes de la β-ciclodextrina con el fin de obtener complejos de inclusión mediante el método de co-precipitación, utilizando N,N-dimetilformamida como disolvente. Los productos de inclusión obtenidos fueron caracterizados mediante espectroscopia IR-ATR, Raman, UV-vis, RMN 1H, RMN 13C, DRX, TGA-DSC, análisis elemental y complexometría con EDTA. Se realizaron pruebas de actividad antibacteriana empleando 6 cepas ATTC (S. aureus ATCC 25923, S. aureus ATCC 29213, E. coli ATCC 25922, P. aeruginosa ATCC 27853, S. Typhimurium ATCC 14028 y K. pneumoniae ATCC BAA-2146) mediante el método de microdilución con caldo Mueller-Hinton; los resultados de actividad biológica para los complejos lantánidos permitieron evidenciar el efecto sinérgico entre el catión lantánido y el ligando cinamato. Igualmente, para los complejos de inclusión se observó una disminución de la concentración mínima inhibitoria (CMI) respecto a los complejos lantánidos iniciales. Los resultados obtenidos con el ADN de timo de ternera y el ADN plasmídico pBR322 permiten proponer una interacción electrostática entre los complejos evaluados y la estructura molecular del ADN.

Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique

Koumiss is a popular dairy product in many lands, traditionally prepared from mare milk with spontaneous fermentation. Mare milk and its fermented derivates are more expensive than cow milk and its fermented derivates, and the possibility exists for producers and dealers to adulterate equine products with bovine items. In this work, we described the development of a triplex real-time PCR based on species-specific TaqMan probes for identification of bovine and equine DNA in milks and dairy products. In addition, a novel designed endogenous control was simultaneously amplified to eliminate possible false negatives. With this methodology, bovine and equine DNA were specifically identified by employing developed primers and probes. The limits of detection of this method were 0.001 ng for cow milk, yogurt, and mare milk, and 0.005 ng for sour soup and koumiss, respectively. In addition, the triplex real-time PCR assay for authentication of animal-derived products was effectively validated using binary DNA and milk mixtures, exhibiting well in terms of specificity, sensitivity, and reproducibility. In short, the triplex PCR assay was verified to be a time-saving and money-saving technique for the identification of bovine and equine DNA in milks and dairy products.

Effect of single-nucleotide polymorphisms on the breeding value of fertility and breeding value of beef in Hungarian Simmental cattle.

The objective of this study was to estimate the effect of single-nucleotide polymorphisms (SNPs) on the breeding value of fertility (BVF) and the breeding value of beef (BVB) in Hungarian Simmental cattle. Genotypes were determined on a high-density Illumina Bovine DNA Chip. Data screening and data identification were performed by multi-locus mixed-model. Statistical analyses were carried out to find associations between individual genotypes and the investigated quality values. Three loci showed considerable association with BVF (-log10 P = 9.5, 9.9 and 14.5, respectively) on chromosomes 9, 28 and 29, respectively. The frequencies of their minor alleles (MAF) were 0.375, 0.355 and 0.354, respectively. Two loci showed association with BVB (-log10 P = 25.3 and 22.7) on chromosomes 2 and 11, respectively (their MAF were 0.438 and 0.229). The abovementioned loci provide a straightforward possibility to assist selection by molecular tools.

Triphenylamine-Appended Half-Sandwich Iridium(III) Complexes and Their Biological Applications.

Organometallic half-sandwich IrIII complexes of the type [(η5 -Cpx )Ir(N^N)Cl]PF6 (Cpx : Cp* or its phenyl Cpxph or biphenyl Cpxbiph derivatives; N^N: triphenylamine (TPA)-substituted bipyridyl ligand groups) were synthesized and characterized. The complexes showed excellent bovine serum albumin (BSA) and DNA binding properties and were able to oxidize NADH to NAD+ (NAD=nicotinamide adenine dinucleotide) efficiently. The complexes induced apoptosis effectively and led to the emergence of reactive oxygen species (ROS) in cells. All complexes showed potent cytotoxicity with IC50 values ranging from 1.5 to 7.1 μm toward A549 human lung cancer cells after 24 hours of drug exposure, which is up to 14 times more potent than cisplatin under the same conditions.

Quantitative analysis of Mycoplasma wenyonii and ‘Candidatus Mycoplasma haemobos” infections in cattle using novel gapN-based realtime PCR assays

Hemotrophic mycoplasmas (HMs) are associated with anemia and other disease complexes in a wide range of livestock and wild animals. Two bovine HM species have been identified to date, i.e. Mycoplasma wenyonii and ‘Candidatus Mycoplasma haemobos’. The study aim was to develop quantitative real-time PCR assays (qPCRs) to detect and quantify M. wenyonii and ‘C. M. haemobos’ and to apply these assays to DNA samples extracted from bovine blood collected in Germany (n = 220) from 22 herds. The qPCR assays specific for M. wenyonii and ‘C. M. haemobos’ were designed using the gapN of the respective hemoplasma species as gene target which encodes the NADP-dependent glyceraldehyde 3-phosphate dehydrogenases (GAPN). The sensitivity of both assays was 10 genome equivalents per reaction, corresponding to 2500 genome equivalents per ml blood. No cross-reactivity with non-target bovine HMs. and other bovine pathogens was observed. Bovine HM DNA was detected in 137 samples (62.27%) with 118 samples (53.64%) being positive for ‘C.M. haemobos’ and 19 samples (8.64%) being positive for M. wenyonii. Thereof, 11 animals (5.00%) were co-infected with both bovine HM species. The found herd prevalence for `C. M. haemobos` was 100.00%, and for M. wenyonii 36.36% with mean bacterial loads of 3.7 × 107 `C. M. haemobos`/mL blood and of 4.29 × 105M. wenyonii/mL blood respectively. Clinical and economic relevance of bovine HM species should be goal of future studies for which the novel gapN qPCR assays can serve as a valuable diagnostic tool.

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs.

Effect of single nucleotide polymorphisms on intramuscular fat content in Hungarian Simmental cattle.

ObjectiveTo estimate effect of single nucleotide polymorphisms on the intramuscular fat content (IMF) of Hungarian Simmental bulls.MethodsGenotypes were determined on high-density Illumina Bovine DNA Chip. After slaughtering of animals, chemical percentage of intramuscular fat was determined from longissimus dorsi muscle. A multi-locus mixed-model was applied for statistical analyses.ResultsAnalyses revealed four loci (rs43284251, rs109210955, rs41630030, and rs41642251) to be highly associated (−log10P>12) with IMF located on chromosome 1, 6, 13, and 17, respectively. The frequency of their minor alleles was 0.426, 0.221, 0.162, and 0.106.ConclusionThe loci above can be useful in selection programs and gives the possibility to assist selection by molecular tools.

Multiplex PCR to discriminate bovine, porcine, and fish DNA in gelatin and confectionery products

Gelatin is widely used in many confectionery products such as gummy, candies, pastilles and marshmallows. However, the acceptance of gelatin in consumer goods largely depends on its animal origins. The main sources of gelatin are pig, cow and fish collagens that have differential acceptance in Halal, Kosher and Hindu foods. Currently, several simplex and duplex PCR assays are available for the determination of gelatin origins but those methods are not only time-consuming and costly but also cannot reveal all information in a single assay platform. For the first time, we documented here a tetraplex PCR assay involving eukaryotic, fish, bovine, and porcine control for the simultaneous detection of these species by a single, easy and reliable approach. The assay was optimized under pure and mixed gelatin matrices and validated by PCR product sequencing. Finally, 38 halal branded confectionery items were screened and 33, 2 and 3 of them yielded positive bands for bovine, porcine and eukaryotes, respectively. The detection limit was 0.1 to 0.001 ng DNA extracted from pure and mixed gelatins. The study is potentially useful in monitoring and certifying halal, kosher and vegetarian issues in processed foods, pharmaceuticals and cosmetics.

Detection of bovine Deltapapillomavirus DNA in peripheral blood of healthy sheep (Ovis aries).

Blood samples from 65 sheep were tested for the presence of bovine Deltapapillomavirus (δPVs) DNA. The sheep were divided into three groups. Sheep in groups 1 and 2 were from Sardinia and Campania, respectively, and were in contact with cattle and grazed on lands contaminated with bracken fern. Sheep in Group 3 lived in closed pens and had no contact with cattle. These sheep were fed hay that did not contain bracken fern. Bovine δPV E5 DNA was detected in blood from 24 of 27 (89%) sheep in Group 1. A single bovine δPV type was detected in the blood from nine (33%) sheep, including the detection of bovine δPV-1 DNA in four sheep, bovine δPV-2 in four and δPV-13 in one sheep. Two δPV types were detected in 33% of the sheep, and three bovine δPV types were detected in 22% of the sheep. Bovine δPVs were detected in 17 of 20 (85%) sheep from Group 2. The detection rate by a single δPV type was 40% with just δPV-1 DNA amplified from two, just δPV-2 DNA from four, and just δPV-13 DNA from two sheep. Two and three δPVs were detected in 30% and 15%, respectively. All sequenced amplicons showed a 100% identity with papillomaviral E5 DNA deposited in GenBank. Bovine δPV-14 DNA sequences were not detected from any sheep. No bovine δPV DNA was revealed in blood samples from sheep in Group 3. The detection of bovine δPV DNA in the blood of sheep means that sheep may be able to be infected by these PVs. This suggests that bovine δPVs could potentially be a previously unrecognized cause of disease in sheep. Furthermore, it is possible that sheep could act as a reservoir for these viruses.

Assessment of milk quality using novel mutations of B2M gene in bovine DNA from milk

ABSTRACTThis study investigated the expression of novel mutations of the β2-microglobulin (B2M) gene in bovine genomic DNA isolated from milk-borne leukocytes and tested the association of these mutations with milk quality traits in Holstein cows. DNA was extracted using a novel protocol allowing for the amplification of long fragments of DNA isolated from small amounts of unprocessed milk samples. Genomic mutations at the B2M locus were explored by DNA sequencing using specific primers and a cluster of five identified within a 729-bp region of intron 3. Specifically, mutations at 7008(G>T), 7202(A>G), 7432(G>T) and 7437(G>C) were all found to be significantly associated with milk protein percentage (P < 0.05), and a mutation at 7026(G>A) was significantly associated with somatic cell count (P < 0.05). The potential to use these mutations as further tools for the pre-evaluation of milk quality is discussed.

New tool application in veterinary forensic science to analyze genetic fingerprinting data

In the last decades legal and other cases in which forensic veterinary science is required are increasing. The principles of investigation follow those used in human beings, although in animal species familial relationship identification is a hard task, due to high level of inbreeding. Familias 3 software is commonly used in human forensic laboratories to calculate probabilities and likelihood ratio (LR) in forensic cases. In the present paper we applied the same principles used in humans to standardize, simplify and accelerate the analysis, applying Familias 3 software in animals. Currently short tandem repeats identification is used as an effective method to trace DNA markers in genotyping. The authors used a standardized microsatellite panel recommended by International Society for Animal Genetics (ISAG) and a large allelic frequency Sicilian bovine DNA profiles database previously developed and published (Cosenza 2015). Based on this bovine matrix, the software permits the allelic frequency data calculation for the investigated samples and the data comparison to find the direct parentages and sibling. We analysed the most common veterinary forensic cases by using Familias 3 modules, alone or combined, to identify the proper module for each case. The software analysis was tested on 196 samples. A practical application of such system should be complementary to the conventional traceability, based on traditional labelling (Guhrin, 1994; Egeland, 2000). The impact of this software on the quality of the analysis report is critical to solve illegal inquiry, as much as the wrong calculation and data interpretation can invalidate the DNA profiling.

The bioavailability of different dissolved organic nitrogen compounds for the freshwater algae Raphidocelis subcapitata

Understanding which factors affect the algal bioavailability of dissolved organic nitrogen (DON) compounds in natural surface waters is important for our understanding of nutrient biogeochemistry and water quality management. We used nitrogen uptake kinetics and algal cell yield to characterize the algal bioavailability of 22 dissolved DON compounds that are commonly found in natural surface waters and wastewater treatment plant effluents, including urea, amino acids, amino sugars, nucleotides, pyrimidines, oraganonitriles, polyacrylamide, EDTA, caffeine, phenolic compounds and humic acids. Twelve of these compounds were highly bioavailable, including urea, dissolved free amino acids, bovine serum albumin, DNA, RNA, ATP, AMP, acetonitrile and caffeine. Four compounds had intermediate bioavailability including two humic acids (Elliott Soil and Pahokee Peat), glycylglycine, RNA and uracil. The remaining six compounds were classified as recalcitrant, i.e., EDTA, 2,3-Dinitrophenol, aminobenzoic acid, polyacrylamide and Aldrich humic acid. For many of the compounds tested, the algal cell yield was only 60–80% of expected relative to DON uptake. These results help explain why some DON compounds are more likely to persist in natural systems, and why the DON pool is often recalcitrant in surface waters.

Influence of resveratrol pretreatment on thawed bovine embryo quality and mitochondrial DNA copy number

This study addresses the hypothesis that SIRT1 upregulation prior to cryopreservation helps in recuperation from cryoinjury and improves the embryo quality. Bovine blastocysts were produced in vitro, and at Day 6 or 7 after fertilization, the blastocysts were cultured with 0 or 1 μM resveratrol for 6 or 24 h. This short duration of resveratrol treatment did not affect the embryo development or the grade of the blastocysts. However, both the durations of resveratrol treatment (6 h or 24 h) significantly increased the expression levels of SIRT1. When embryos pre-treated with resveratrol (0 or 1 μM) were cryopreserved and subsequently thawed, the hatching rates following 48 h of incubation were significantly higher for the resveratrol-treated embryos than for vehicle-treated embryos. Moreover, the resveratrol pretreatment significantly increased the copy number of mitochondrial DNA in the embryos, irrespective of the treatment durations. The in vitro-produced embryos at 6 days of insemination and the in vivo-developed embryos, which were collected from the donor cows at 6.5 days of insemination, were treated with resveratrol for 6 or 24 h prior to cryopreservation, respectively. The resveratrol pretreatment (for 6 or 24 h) resulted in high conception rate after thawing and transfer to the recipients, in both the in vivo and in vitro-produced embryos. In conclusion, our results suggest that pretreatment of bovine embryos with resveratrol improves the quality of embryos after cryopreservation and thawing probably through mitochondrial synthesis.

Authentication of caprine milk and cheese by commercial qPCR assay

The objective of the study was to investigate potential adulteration of commercial caprine milks and cheeses with bovine milk using commercial qPCR assay. The assay comprised of bovine-, ovine- and caprine-specific primers and TaqMan probe and mammalian internal control. Specificity, sensitivity, linearity, reproducibility and efficiency of the bovine assay were tested as well. Specificity was verified by running reaction on the DNA of other milk-producing species (caprine and ovine) and made-up bovine-caprine (v/v) milk mixes. In both experiments, a bovine DNA fragment was amplified whereas no amplification was obtained from the other species. Sensitivity, linearity, reproducibility and efficiency were tested on 10-fold dilution series of 10 ng bovine DNA. The assay has shown good linearity (R2 = 0.983) within whole range, with efficiency of 86% and excellent reproducibility (SD around the CT for the technical replicates &lt;0.5). The sensitivity was adequate, as calculated LOD and LOQ were 1.44 pg and 2.94 pg of bovine DNA, respectively. Finally, the assay was used to authenticate 5 caprine milk samples and 5 caprine cheese samples, purchased from local supermarkets. Totally, 1 milk sample has shown the fluorescence signal, which exceeded baseline in cycle 39.01 ±0.69. However, the signal was above LOD and LOQ suggesting that there could not be unambiguously declared any adulteration with bovine milk. Amplification of bovine-specific DNA was not observed in the other samples indicating products were not adulterated. The commercial qPCR assay has proved that real-time PCR assays, as well as DNA-based techniques in a general, are the excellent and reliable tools for fighting with frauds in the food industry and protecting the public health.

Bovine leukaemia virus DNA in fresh milk and raw beef for human consumption.

Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leucosis, which has been reported worldwide. BLV has been found recently in human tissue and it could have a significant impact on human health. A possible hypothesis regarding viral entry to humans is through the consumption of infected foodstuffs. This study was aimed at detecting the presence of BLV DNA in raw beef and fresh milk for human consumption. Nested PCR directed at the BLV gag gene (272 bp) was used as a diagnostic test. PCR products were confirmed by Sanger sequencing. Forty-nine per cent of the samples proved positive for the presence of proviral DNA. This is the first study highlighting the presence of the BLV gag gene in meat products for human consumption and confirms the presence of the viral DNA in raw milk, as in previous reports. The presence of viral DNA in food products could suggest that viral particles may also be found. Further studies are needed to confirm the presence of infected viral particles, even though the present findings could represent a first approach to BLV transmission to humans through foodstuff consumption.

Ultrasensitive Detection of Staphylococcus aureus and Listeria monocytogenes Genomic DNA by Nanoparticle‐Enhanced Surface Plasmon Resonance Imaging

AbstractThe rapid, sensitive and specific detection of pathogenic bacteria is key to the prevention of a variety of foodborne illnesses representing a significant threat to public health. Conventional methods for the detection and identification of pathogens may take up to a few days to yield an answer, and for this reason, efforts have been made to develop new biosensing platforms for the efficient detection of foodborne pathogens. Here we report the use of a nanoparticle‐enhanced surface plasmon resonance (SPR) imaging biosensor for the rapid and femtomolar‐attomolar detection of Staphylococcus aureus and Listeria monocytogenes genomic DNAs. We performed the nanoparticle enhanced SPR imaging detection of pathogen genomic DNAs with no need for the polymerase chain reaction (PCR) pre‐amplification of the sequences to be detected. Here we show that peptide nucleic acid (PNA) probes can discriminate between two pathogenic genomic DNAs. We tested the selectivity of the detection method against control probe sequences or contaminating bovine genomic DNA and demonstrated, for the first time, the attomolar detection of Staphylococcus aureus genomic DNA in the presence of a 10‐fold higher concentration of bovine genomic DNA.

Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed.

Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures. The specificity of the ddPCR assays was confirmed by testing both target species and additional 18 non-target species. Linear regression established a detection range between 79 and 33200 copies of the target molecule from 0.26 to 176 pg of fresh animal tissue DNA with a coefficient of determination (R2) of 0.997–0.999. The quantification ranges of the methods for testing fortified heat-processed food and feed samples were 0.05–3.0% (wt/wt) for the bovine and turkey targets, and 0.01–1.0% (wt/wt) for pork and chicken targets. Our methods demonstrated acceptable repeatability and reproducibility for the analytical process for food and feed samples. Internal validation of the PCR process was monitored using a control chart for 74 consecutive ddPCR runs for quantifying bovine DNA. A matrix effect was observed while establishing calibration curves with the matrix type under testing, and the inclusion of an internal control in DNA extraction provides a useful means to overcome this effect. DNA degradation caused by heating, sonication or Taq I restriction enzyme digestion was found to reduce ddPCR readings by as much as 4.5 fold. The results illustrated the applicability of the methods to quantify meat species in food and feed samples without the need for a standard curve, and to potentially support enforcement activities for food authentication and feed control. Standard reference materials matching typical manufacturing processes are needed for future validation of ddPCR assays for absolute quantification of meat species.